Methods for single molecule sequencing

ABSTRACT

Provided herein are methods and systems for sequencing a single nucleic acid molecule utilizing a polymerase enzyme, a template nucleic acid, and a polymerase reagent solution.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/435,166 filed Dec. 16, 2016, which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The invention relates to methods for single molecule nucleic acidsequencing.

INTRODUCTION

Current sequencing technologies can be grouped into two main categories:short-read sequencing and long-read sequencing. In each category, DNA iscleaved into pieces with lengths up to a certain number of nucleotidesor basepairs (bp). In all cases, all pieces of DNA are spread into a 2dimensional array and are detected by a sensor array corresponding towhere at least one sensor is matched with a piece of DNA.

Short-read sequencing approaches are simple cycle based technologiesthat includes sequencing-by-ligation (SBL) and sequencing-by-synthesis(SBS). SBL approaches includes SOLID (Thermo Fisher) and CompleteGenomics (BGI). With SOLID, read lengths around 75 basepairs (bps) isreached while with Complete Genomics approach 28 to 100 basepair readsare feasible. With these approaches structural variation and genomeassembly is not possible and they are susceptible to homopolymer errors.Their runtimes are on the order of several days. Illumina and Qiagen'sGeneReader technology use SBS approach with Cyclic ReversibleTermination. They can reach up to 300 bp. However, a major drawback isunder representation of AT and GC rich regions, substation errors andhigh half positive rate. On the other hand, other SBS approaches such as454 pyrosequencing and Ion Torrent (Thermo Fisher) use single-nucleotideAddition/Termination. 454 pyrosequencing could reach 400 bp while IonTorrent can achieve 700 bp read lengths. However, although thesetechnologies are faster and good for point of care, they also have manydrawbacks including domination of insertion/deletion errors, andhomopolymer region errors. They cannot be used to reveal long-rangegenomic or transcriptomic structure, and cannot do paired endsequencing.

Long-read sequencing approaches include two main types, syntheticlong-read sequencing or real-time long-read sequencing. Syntheticlong-read sequencing used by Illumina and 10X Genomics focuses onlibrary preparation that leverages barcodes and allows computationalassembly of large fragments. In fact, these technologies do not doactual long-reads, rather they do short-reads, in which the DNA piecesare organized using a barcoding approach, which helps eliminate somecomplexity during analysis, which allows obtaining data similar toactual long-read methods. However, this approach has a very high costdue, in part, to its requiring even more coverage. The other type oflong-read sequencing is real-time long-read sequencing, which has beenused by Pacific Biosciences and Oxford Nanopore Technologies. Unlikesynthetic long-read sequencing, real-time long-read sequencing does notrely on clonal population of amplified DNA and does not require chemicalcycling. Nanopore's technology has very high error rates around 30%,which also require very high coverage that contributes significantly tothe cost. Using modified bases has also been particularly challengingfor Nanopore's technology, which has generated unique signals that makesthe analysis even more complex. Pacific Biosciences can reach readlengths up to 4000-5000 bps. However, due to high single-pass errorrates around 15% for long reads, high coverage is required, which makes1 Gb sequencing cost more than $1000 (see., e.g., Goodwin et al., Nat.Rev. Genet. 17:333-351; 2016).

Since, a large majority of current technologies offer short read lengths(around 40-100 bases long) of nucleotides per unit, one of the mostchallenging problem lies in alignment of small pieces of sequences intoone large meaningful sequence, and analyzing high coverage data and thepost-processing of the loads of generated data with complicatedalgorithms using powerful super computers. Newer generation singlemolecule based sequencing technologies can potentially address thisissue. However, each of these prior art technologies have high errorrates requiring high coverages (multiple reads of the same region of asequence) often around 30× to 100× in order to obtain a reliable data.

Accordingly, there is a need for improved methods for single moleculenucleic acid sequencing.

SUMMARY

Provided herein are methods for sequencing a nucleic acid templatecomprising:

providing a sequencing mixture comprising (i) a polymerase enzyme, (ii)a template nucleic acid, and (iii) a polymerase reagent solution havingthe components for carrying out template directed synthesis of a growingnucleic acid strand, wherein said polymerase reagent solution includes acomponent for a requenching reaction (e.g., a quenching enzyme, and thelike) and a plurality of types of nucleotide analogs; wherein each typeof nucleotide analog has a labeled leaving group that is cleavable bythe polymerase, and each type of nucleotide analog has a differentlabel, wherein the labeled leaving group is cleaved uponpolymerase-dependent binding of a respective nucleotide analog to thetemplate strand;

carrying out nucleic acid synthesis such that a plurality of nucleotideanalogs are added sequentially to the template whereby: a) a nucleotideanalog associates with the polymerase, b) the nucleotide analog isincorporated on the template strand by the polymerase when the labeledleaving group on that nucleotide analog is cleaved by the polymerase,wherein the labeled leaving group emits light upon cleavage, then c) thelabeled leaving group on the nucleotide analog is quenched by therequenching reaction; and

detecting light from the labels while nucleic acid synthesis isoccurring, and using light detected in the time between step b) when thelabelled leaving group is cleaved, and step c) in which the labeledleaving group is quenched, to determine a sequence of the templatenucleic acid.

The invention methods are useful for a variety of uses including wholegenome sequencing and SNP-variant detection.

The disclosed invention is a single molecule sequencing technology basedon monitoring individual polymerase enzymes as they incorporate dNTPssequentially. In a particular embodiment, the invention encompasses aprocess where each time polymerase incorporates a dNTPs complementary tothe template, a fluorescence signal is generated during theincorporation process and subsequently is quenched once theincorporation process ends. The process repeats for the next dNTPincorporation (FIG. 1).

More particularly, each time a polymerase incorporates a modifieddeoxyribonuleoside triphosphate (dNTP) nucleotide analog to the strandcomplementary to the template DNA, a fluorescence signal specific to thetype of the nucleotide attached is generated. There are four types ofdNTPs, namely deoxyadenosine triphosphate (dATP), deoxyguanosinetriphosphate (dGTP), deoxycytidine triphosphate (dCTP), anddeoxythymidine triphosphate (dTTP). Each nucleotide generates a uniquefluorescence signal (e.g., in red, yellow, green, or blue, and the like)while they are being attached to the complementary strand by thepolymerase enzyme. Upon the completion of attachment of the nucleotideanalog to the 3′ moiety of the previously attached nucleotide analog,the fluorescence generated by the leaving group is detected by anappropriate fluorescence sensor and/or detection device and then it issubsequently rapidly quenched (FIG. 1).

Sequencing is achieved by detecting the fluorescence generated each timea nucleotide is added to the complementary strand revealing the type ofnucleotide. Therefore, each specific nucleotide attachment generates ashort peak of a fluorescence signal that can be detected by afluorescence sensor. As a result a data array of succeeding, sequentialcolors is produced, which can be converted into a corresponding dataarray of nucleotide sequence (FIG. 1).

An advantage provided by the invention methods disclosed herein lies inits simplicity and innovative chemistry that significantly reducesbackground signal during detection thereby improving sensitivity. Inaccordance with the present invention methods, less modification of thereaction conditions involving reagents and enzymes improves specificity,efficiency and rate. Also in accordance with the present inventionmethods, polymerase operates in near ideal conditions, and iscontemplated to reach very long read lengths around tens of thousands ofbases per DNA polymerase molecule by utilizing high sensitivity andspecificity together with requiring significantly less post-processingand analysis of the data produced. The combined features of theinvention methods disclosed herein reduces the cost both for therespective devices and each run, while achieving high specificity inaddition to decreasing the time per test considerably compared tocompeting technologies. Accordingly, the disclosed invention methods andsystems allow realization of very low cost and real-time sequencingsystems without adversely affecting specificity.

INCORPORATION BY REFERENCE

Herein, all issued patents, published patent applications, andnon-patent publications that are mentioned in this specification areherein incorporated by reference in their entirety for all purposes, tothe same extent as if each individual issued patent, published patentapplication, or non-patent publication were specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a general illustration of one embodiment of the inventionsequencing method: DNA Polymerase uses modified dNTPs with initiallyquenched fluorophores as building blocks. Upon binding to polymerase,the fluorescent molecule become activated and later it is cleaved off,detected, and finally is quenched.

FIG. 2A shows a depiction of a fluorophore attached to the terminalphosphate of a dNTP, which is quenched by the respective nucleobasewhile the fluorophore is attached. Each respective nucleobase has adifferent quenching capability of the different fluorophores.

FIG. 2B shows the polymerase-dependent binding of a respectivenucleotide analog having a fluorophore attached therein to the templatestrand and the cleaving of the labeled pyrophosphate that has thefluorophore attached, which causes the fluorophore to emit afluorescence light signal.

FIG. 2C further shows that during the nucleotide analog dNTP interactionwith polymerase, fluorescence is generated upon cleavage of the labeledpyrophosphate generating a fluorescence signal corresponding to thecolor of the respective fluorophore. There is a unique coloredfluorphore for each class of nucleotide analog dNTPs, such that eachtype of nucleotide analog has a different label.

FIG. 2D shows that after the labeled pyrophosphate is released from itsrespective dNTP, it next interacts with ATP sulfurylase, which binds therespective labeled pyrophosphate to to adenosine 5′-phosphosulfate (APS)resulting in quenching of the fluorophore by adenine upon its binding,thereby substantially reducing fluorescence background noise.

FIG. 3 shows the dual-quenching chemisty of an embodiment of theinvention sequencing method that employs dual-quenching of an attachedfluorophore, whereby the fluorophore is quenched by the respectivenucleobase and an additional non-covalently bound quencher (A.) Stage 1:dNTP incorporation by polymerase and fluorescence generation viafluorescent labeled pyrophosphate release. (B.) Stage 2: Quenching ofthe released fluorescent pyrophosphate.

FIG. 4 shows a simplified schematic of the biochemical process of dNTPincorporation into a template strand.

DETAILED DESCRIPTION

Provided herein are methods for sequencing a nucleic acid templatecomprising:

providing a sequencing mixture comprising (i) a polymerase enzyme, (ii)a template nucleic acid, and (iii) a polymerase reagent solution havingthe components for carrying out template directed synthesis of a growingnucleic acid strand, wherein said polymerase reagent solution includes acomponent for a requenching reaction (e.g., a quenching enzyme, and thelike) and a plurality of types of nucleotide analogs; wherein each typeof nucleotide analog has a labeled leaving group that is cleavable bythe polymerase, and each type of nucleotide analog has a differentlabel, wherein the labeled leaving group is cleaved uponpolymerase-dependent binding of a respective nucleotide analog to thetemplate strand;

carrying out nucleic acid synthesis such that a plurality of nucleotideanalogs are added sequentially to the template whereby: a) a nucleotideanalog associates with the polymerase, b) the nucleotide analog isincorporated on the template strand by the polymerase when the labeledleaving group on that nucleotide analog is cleaved by the polymerase,wherein the labeled leaving group emits light upon cleavage, then c) thelabeled leaving group on the nucleotide analog is quenched by therequenching reaction; and

detecting light from the labels while nucleic acid synthesis isoccurring, and using light detected in the time between step b) when thelabelled leaving group is cleaved, and step c) in which the labeledleaving group is quenched, to determine a sequence of the templatenucleic acid.

As used herein a “polymerase enzyme” refers to the well known proteinresponsible for carrying out nucleic acid synthesis. A preferredpolymerase enzyme for use herein is a DNA polymerase. In naturalpolymerase mediated nucleic acid synthesis, a complex is formed betweena polymerase enzyme, a template nucleic acid sequence, and a primingsequence that serves as the point of initiation of the syntheticprocess. During synthesis, the polymerase samples nucleotide monomersfrom the reaction mix to determine their complementarity to the nextbase in the template sequence. When the sampled base is complementary tothe next base, it is incorporated into the growing nascent strand. Thisprocess continues along the length of the template sequence toeffectively duplicate that template. Although described in a simplifiedschematic fashion, the actual biochemical process of incorporation canbe relatively complex. A diagrammatical representation of theincorporation biochemistry is provided in FIG. 4. This diagram is not acomplete description of the mechanism of nucleotide incorporation.During the reaction process, the polymerase enzyme undergoes a series ofconformational changes which can be essential steps in the mechanism.

As shown in FIG. 4, the synthesis process begins with the binding of theprimed nucleic acid template (D) to the polymerase (P) at step 2.Nucleotide (N) binding with the complex occurs at step 4. Step 6represents the isomerization of the polymerase from the open to closedconformation. Step 8 is the chemistry step in which the nucleotide isincorporated into the growing strand. At step 10, polymeraseisomerization occurs from the closed to the open position. Thepolyphosphate component that is cleaved upon incorporation is releasedfrom the complex at step 12. While the figure shows the release ofpyrophosphate, it is understood that when a labeled nucleotide ornucleotide analog is used, the component released may be different thanpyrophosphate. In many cases, the systems and methods of the inventionuse a nucleotide analog having a label on its terminal phosphate, suchthat the released component comprises a polyphosphate connected to a dye(e.g., a label pyrophosphate; PP,). With a natural nucleotide ornucleotide analog substrate, the polymerase then translocates on thetemplate at step 14. After translocation, the polymerase is in theposition to add another nucleotide and continue around the reactioncycle.

Preferred polymerase enzymes for use herein include DNA polymerases,which can be classified into six main groups based upon variousphylogenetic relationships, e.g., with E. coil Pol I (class A), E. coliPol II (class B), E. coil Pol III (class C), Euryarchaeotic Pol II(class D), human Pol beta (class X), and E. coil UmuC/DinB andeukaryotic RAD30/xeroderrna pigmentosum variant (class Y). For a reviewof nomenclature, see, e.g., Burgers et al. (2001) “Eukaryotic DNApolymerases: proposal for a revised nomenclature” J Biol Chem.276(47):43487-90. For a review of polymerases, see, e.g., Hubscher etal. (2002) “Eukaryotic DNA Polymerases” Annual Review of BiochemistryVol. 71: 133-163; Alba (2001) “Protein Family Review: Replicative DNAPolymerases” Genome Biology 2(1):reviews 3002.1-3002.4; and Steitz(1999) “DNA polymerases: structural diversity and common mechanisms” JBiol Chem 274:17395-17398; each of which are incorporated herein byreference in their entirety. The basic mechanisms of action for manypolymerases have been determined. The sequences of literally hundreds ofpolymerases are publicly available, and the crystal structures for manyof these have been determined, or can be inferred based upon similarityto solved crystal structures for homologous polymerases.

Many such polymerases suitable for nucleic acid sequencing are readilyavailable. For example, human DNA Polymerase Beta is available from R&Dsystems. Preferred DNA polymerase for use herein, include DNA polymeraseI that is available from Epicenter, GE Health Care, Invitrogen, NewEngland Biolabs, Promega, Roche Applied Science, Sigma Aldrich and manyothers. The Klenow fragment of DNA Polymerase I is available in bothrecombinant and protease digested versions, from, e.g., Ambion, Chimerx,eEnzyme LLC, GE Health Care, Invitrogen, New England Biolabs, Promega,Roche Applied Science, Sigma Aldrich and many others. PHI.29 DNApolymerase is available from e.g., Epicentre. Poly A polymerase, reversetranscriptase, Sequenase, SP6 DNA polymerase, T4 DNA polymerase, T7 DNApolymerase, and a variety of thermostable DNA polymerases (Taq, hotstart, titanium Taq, etc.) are available from a variety of these andother sources. Other commercial DNA polymerases include PhusionhMHigh-Fidelity DNA Polymerase, available from New England Biolabs;GoTaq.R™. Flexi DNA Polymerase, available from Promega; RepIiPHI.™..PHI.29 DNA Polymerase, available from Epicentre Biotechnologies;PfuUltra.™. Hotstart DNA Polymerase, available from Stratagene; KOD HiFiDNA Polymerase, available from Novagen; and many others.

Available DNA polymerase enzymes have also been modified in any of avariety of ways, e.g., to reduce or eliminate exonuclease activities(many native DNA polymerases have a proof-reading exonuclease functionthat interferes with, e.g., sequencing applications), to simplifyproduction by making protease digested enzyme fragments such as theKlenow fragment recombinant, etc. As noted, polymerases have also beenmodified to confer improvements in specificity, processivity, andimproved retention time of labeled nucleotides inpolymerase-DNA-nucleotide complexes (e.g., WO 2007/076057 POLYMERASESFOR NUCLEOTIDE ANALOGUE INCORPORATION by Hanzel et al. and WO2008/051530 POLYMERASE ENZYMES AND REAGENTS FOR ENHANCED NUCLEIC ACIDSEQUENCING by Rank et al.), to alter branch fraction and translocation(e.g., U.S. patent application Ser. No. 12/584,481 filed Sep. 4, 2009,by Pranav Patel et al. entitled “ENGINEERING POLYMERASES AND REACTIONCONDITIONS FOR MODIFIED INCORPORATION PROPERTIES”), to increasephotostability (e.g., U.S. patent application Ser. No. 12/384,110 filedMar. 30, 2009, by Keith Bjornson et al. entitled “Enzymes Resistant toPhotodamage”), and to improve surface-immobilized enzyme activities(e.g., WO 2007/075987 ACTIVE SURFACE COUPLED POLYMERASES by Hanzel etal. and WO 2007/076057 PROTEIN ENGINEERING STRATEGIES TO OPTIMIZEACTIVITY OF SURFACE ATTACHED PROTEINS by Hanzel et al.). Any of theseavailable polymerases can be modified in accordance with the inventionto decrease branching fraction formation, improve stability of theclosed polymerase-DNA complex, and/or alter reaction rate constants.

DNA polymerases that are preferred substrates for mutation to decreasebranching fraction, increase closed complex stability, or alter reactionrate constants include Taq polymerases, exonuclease deficient Taqpolymerases, E. coil DNA Polymerase 1, Klenow fragment, reversetranscriptases, PHI-29 related polymerases including wild type PHI-29polymerase and derivatives of such polymerases such as exonucleasedeficient forms, T7 DNA polymerase, T5 DNA polymerase, an RB69polymerase, etc.

In addition, the polymerases can be further modified forapplication-specific reasons, such as to increase photostability, e.g.,as taught in U.S. patent application Ser. No. 12/384,110 filed Mar. 30,2009, to improve activity of the enzyme when bound to a surface, astaught, e.g., in WO 2007/075987, and WO 2007/076057, or to includepurification or handling tags as is taught in the cited references andas is common in the art. Similarly, the modified polymerases describedherein can be employed in combination with other strategies to improvepolymerase performance, for example, reaction conditions for controllingpolymerase rate constants such as taught in U.S. patent application Ser.No. 12/414,191 filed Mar. 30, 2009, and entitled “Two slow-steppolymerase enzyme systems and methods,” incorporated herein by referencein its entirety for all purposes.

As used herein, the phrase “template nucleic acid” refers to anysuitable polynucleotide, including double-stranded DNA, single-strandedDNA, single-stranded DNA hairpins, DNA/RNA hybrids, RNAs with arecognition site for binding of the polymerizing agent, and RNAhairpins. Further, target polynucleotides suitable as template nucleicacids for use in the invention sequencing methods may be a specificportion of a genome of a cell, such as an intron, regulatory region,allele, variant or mutation; the whole genome; or any portion thereof.In other embodiments, the target polynucleotides may be mRNA, tRNA,rRNA, ribozymes, antisense RNA or RNAi. The target polynucleotide may beof any length, such as at between about 10 bases up to about 100,000bases, between about 10,000 bases up to about 90,000 bases, betweenabout 20,000 bases up to about 80,000 bases, between about 30,000 basesup to about 70,000 bases, between about 40,000 bases up to about 60,000bases, or longer, with a typical range being between about 10,000-50,000bases. Also contemplated herein are target template nucleic acid lengthsof between about 100 bases and 10,000 bases.

The template nucleic acids of the invention can also include unnaturalnucleic acids such as PNAs, modified oligonucleotides (e.g.,oligonucleotides comprising nucleotides that are not typical tobiological RNA or DNA, such as 2′-O-methylated oligonucleotides),modified phosphate backbones and the like. A nucleic acid can be e.g.,single-stranded or double-stranded.

As used herein, the phrase “nucleotide analog” refers to modifiednucleotides that can be used in DNA synthesis (e.g., modified dNTPs suchdATP, dTTP, dGTP, dCTP and dUTP). The nucleotide analogs for use in theinvention can be any suitable nucleotide analog that is capable of beinga substrate for the polymerase and for the selective cleaving activity.It has been shown that nucleotides can be modified and still used assubstrates for polymerases and other enzymes. Where a variant of anucleotide analog is contemplated, the compatibility of the nucleotideanalog with the polymerase or with another enzyme activity such asexonuclease activity can be determined by activity assays. The carryingout of activity assays is straightforward and well known in the art.

The nucleotide analog can be, for example, a nucleoside polyphosphatehaving three or more phosphates in its polyphosphate chain with a labelon the portion of the polyphosphate chain that is cleaved uponincorporation into the growing strand. The polyphosphate can be a purepolyphosphate, e.g. —O—PO3- or a pyrophosphate (e.g., PP_(i)), or thepolyphosphate can include substitutions. Additional details regardinganalogs and methods of making such analogs can be found in U.S. Pat.Nos. 7,405,281; 9,464,107, and the like; incorporated herein byreference in its entirety for all purposes.

Alternative labeling strategies may employ inorganic materials aslabeling moieties, such as fluorescent or luminescent nanoparticles,e.g. nanocrystals, i.e. Quantum Dots, that possess inherent fluorescentcapabilities due to their semiconductor make up and size in thenanoscale regime (See, e.g., U.S. Pat. Nos. 6,861,155, 6,699,723,7,235,361, which are incorporated by reference herein for all purposes).Such nanocrystal materials are generally commercially available from,e.g., Life Technologies, (Carlsbad Calif.). Again, such compounds may bepresent as individual labeling groups or as interactive groups or pairs,e.g., with other inorganic nanocrystals or organic fluorophores. In somecases fluorescent proteins can be used such as green fluorescent protein(GFP, EGFP), blue fluorescent protein (EBFP, EBFP2, Azurite, mKalamal)cyan fluorescent protein (ECFP, Cerulean, CyPet) and yellow fluorescentprotein derivatives (YFP, Citrine, Venus, YPet). Also contemplated foruse herein is fluorescent cell barcoding using multipole fluorescencedyes procuding multiple color coded signals for detection, such asdescribed in Krutzek et al., Curr Protoc Cytom. 2011 January; CHAPTER:Unit-6.31. (doi:10.1002/0471142956.cy0631s55.); which is incorporatedherein by reference in its entirety for all purposes.

In a preferred embodiment, the nucleotide analog is modified by adding afluorophore to a terminal phosphate (see, e.g, Yarbrough et al., J.Biol. Chem., 254:12069-12073, 1979; incorporated herein by reference inits entirety for all purposes), such that when the PP_(i) labeledleaving group is generated by the polymerase when the nucleotide analogis incorporated into the template strand. In this embodiment, thefluorophore is attached in such a way so that the fluorescent signal isquenched by the respective nucleobase as set forth, for example, inSeidal et al, J Phys. Chem., 1996, 100:5541-5553, incorporated herein byreference in its entirety for all purposes. There are four types ofdNTPs, namely deoxyadenosine triphosphate (dATP), deoxyguanosinetriphosphate (dGTP), deoxycytidine triphosphate (dCTP), anddeoxythymidine triphosphate (dTTP). In preferred embodiments of theinvention methods disclosed herein, each respective dNTP is modifiedusing a different, unique fluorophore relative to the other dNTPs, suchthat each time a polymerase incorporates a modified deoxyribonuleosidetriphosphate (dNTP) nucleotide analog to the strand complementary to thetemplate DNA, a fluorescence signal specific to the class or type of thenucleotide (e.g., unique signals for each of dATP, dTTP, dGTP and dCTP)attached is generated.

In another embodiment, to the nucleotide analog that already has aquenched fluorophore therein, stronger quenching in the inventionsequencing methods is achieved by binding an additional removablequencher molecule to that nucleotide analog, wherein the removablequencher molecule is also released (e.g., kicked-out) upon binding withpolymerase. The utilization of this additional second quencher, inaddition to the quenching by the nucleobase within the nucleotideanalog, is referred to herein as dual-quenching of the respectivenucleotide analog. In this particular embodiment of dual-quenching, aremovable quencher can be attached or bound to the nucleotide analogbefore interacting with the polymerase through, for example, weakhydrogen bonding or electrostatic interaction (or the like) with thebase or sugar of the nucleotide analog already having a fluorophoretherein that is quenched by the respective nucleobase as set for above(FIG. 3). The resulting dual-quenching of the nucleotide analogfluorescent signal further enhances the fidelity of the inventionsequencing method. In this embodiment, the quencher on APS (FIG. 2C) maybe covalently attached as well as non-covalently attached. It iscontemplated herein that APS used herein can have extra (e.g., morethan 1) quenchers without adversely affecting the sequencing reaction.

Each nucleotide generates a unique fluorescence signal (e.g., in red,yellow, green, or blue, and the like) while they are being attached tothe complementary strand by the polymerase enzyme. Upon the completionof attachment of the nucleotide analog to the 3′ moiety of thepreviously attached nucleotide analog, the fluorescence generated by theleaving group (e.g., fluorescent pyrophosphate; PPi) is detected by anappropriate fluorescence sensor and/or detection device and then thelabeled pyrophosphate is subsequently rapidly quenched (FIG. 1).

Using the invention methods provided herein, a particular signalindicating the particular type of nucleotide will be generated onlyduring the specific interaction of the nucleotide with the polymerase.The pre- and post- polymerase interaction states will be similar; andthe signal will “change” during the interaction with the polymerase. Forexample, in the fluorescence quenching embodiment described herein:

-   -   1—Initially, there is either no or very low background        fluorescence.    -   2—During polymerase interaction, a specific type of fluorescence        is generated.    -   3—After PP_(i) release and the quenching reaction of        pyrophosphate, the signal goes back to initial state.

In another embodiment employing Plasmonics, the proximity of metalnanoparticles changes the signal: plasmonic shift. For example, the baseor sugar of a nucleotide has a metal nanoparticle attached, and theterminal phosphate has another metal nanoparticle attached. In anotherembodiment, this is also used to identify the respective type of base:via either different respective metals such as gold, silver, copper,aluminum, and the like; or metal particles having different diameterscan be used:

-   -   1—Initially, each nucleotide has two metal nanoparticles that        are coupled (plasmonically), which corresponds to a particular        background plasmonic signal.    -   2—During polymerase interaction, with the release of the metal        nanoparticle with the pyrophosphate, a plasmonic couple is        broken creating a plasmonic shift following the release, which        corresponds to the signal being detected for the respective        nucleotide.    -   3—Afterwards, the released pyrophosphate with metal nanoparticle        is attached to APS such that all the metal nanoparticles return        to their initial coupled state.

In yet another embodiment, Fluorescence Resonance Energy Transfer (FRET)is contemplated for use herein instead of quenching another base signal.For example when utilizing FRET in the invention methods rather than aquenching reaction, there is an acceptor dye (a long wavelength such asred) on the base or sugar. In this embodiment, the fluorophores on theterminal phosphate are donors having shorter wavelengths (e.g., blue,green, yellow, orange), such that when combined they do FRET to theacceptor and we only see red fluorescence, which is the base signal.Then, after attachment and upon cleavage see their specific fluorescenceuntil they are recombined with the secondary reaction to FRET and emitred again. Numerous donor:acceptor FRET pairs are well-known in the artfor use herein. Briefly:

-   -   1—Initially, there is a particular fluorescence emission (e.g.,        “red”).    -   2—During interaction with polymerase, a specific type of        fluorescence is generated different than red (e.g., blue, green,        yellow, orange).    -   3—After, release of pyrophosphate signal goes back to initial        state (“red”).

As used herein, the phrase “labeled leaving group” refers to thepolyphosphate chain having a label, e.g., a fluorophore, or the like,attached therein, that is released from a respective dNTP when and/orupon cleavage by the polymerase enzyme (e.g., DNA pol) during theincorporation of the respective dNTP into the template nucleic acidstrand. In a particular embodiment herein, the polyphosphate is afluorescently labeled pyrophosphate (PPi) that is cleaved and releasedinto the reaction mixture for subsequent fluorescence detection prior tothe labeled pyrophosphate becoming quenched by a component for arequenching reaction (e.g., a quenching enzyme, and the like) as setforth herein (see FIG. 2B).

As used herein, the phrase “polymerase reagent solution” refers to themixture of components necessary for carrying out the template directedsynthesis of a growing nucleic acid. The polymerase reagent solution foruse with a polymerase, e.g., DNA pol I, and the like, includes aquenching enzyme (e.g., ATP sulfurylase, and the like) and suitableconcentrations of dNTPs, e.g., fluorophore-modified nucleotide analogsdescribed herein. In a preferred embodiment, the concentrations of dNTPsemployed are much higher than has been heretofore possible because, inpart, of the low fluorescent background resulting from the labeledleaving groups (e.g., fluorescent pyrophosphate; PP_(i)) advantageouslyemployed in the invention methods. Because the quenching enzyme (e.g.,ATP sulfurylase) and polymerase rates can vary significantly dependingon the type and source of the enzymes, the rate of quenching achieved bythe ATP sulfurylase reaction employed herein can be adjusted separatelyby adjusting reaction conditions such as ATP sulfurylase concentration,and the like as described herein.

As used herein, the phrase “sequencing mixture” refers to the componentsthat are used to carry out the invention single molecule sequencingreactions. In one embodiment, the sequencing mixture includes apolymerase enzyme (e.g., DNA pol I), a template nucleic acid, and apolymerase reagent solution including a component for a requenchingreaction (e.g., a quenching enzyme, such as ATP sulfurylase, and thelike) and labeled nucleotide analogs therein. In accordance with thepresent invention, the sequencing mixture used provides the followingadvantages in the invention sequencing methods over previous sequencingmethods: the polymerase employed functions in its ideal state; there isno need to modify a polymerase enzyme; the use of high nucleotide (e.g.,dNTP) concentrations results in optimum efficiency; requires only lowintensity excitation light, which advantageously reduces photobleachingof the fluorophores and reduces the denaturing of the polymerase enzyme;provides virtually no fluorescent background, which improves specificityand sensitivity of the base calling; does not require sophisticatedoptics or nanostructured chip design, which reduces cost; it provideshigh specificity, which reduces the need for high coverage; and provideslong read lengths (e.g., about 50 Kb to 1 gene/cell) with much lesscomputer processing required relative to prior art methods.

As used herein the phrase “requenching reaction” refers to any reactionthat can requench a signal emitter, such as the released fluorophore inFIGS. 2B and 2C, or any other moiety emitting a signal to be detectedherein. As set forth in the methods herein, the signals correspond to aparticular nucleotide base in the DNA sequence. As used herein, “acomponent for a requenching reaction” can include a quenching enzyme,such as ATP sulfurylase, and the like.

The reaction conditions used can also influence the relative rates ofthe various reactions. Thus, controlling the reaction conditions can beuseful in ensuring that the sequencing method is successful at callingthe bases within the template at a high rate. The reaction conditionsinclude, e.g., the type and concentration of buffer, the pH of thereaction, the temperature, the type and concentration of salts, thepresence of particular additives which influence the kinetics of theenzyme, and the type, concentration, and relative amounts of variouscofactors, including metal cofactors. Manipulation of reactionconditions to achieve or enhance two slow step behavior of polymerasesis described in detail in U.S. Pat. No. 8,133,672, incorporated hereinby reference.

Enzymatic reactions are often run in the presence of a buffer, which isused, in part, to control the pH of the reaction mixture. The type ofbuffer can in some cases influence the kinetics of the polymerasereaction in a way that can lead to two slow-step kinetics, when suchkinetics are desired. For example, in some cases, use of IRIS as bufferis useful for obtaining a two slow-step reaction. Suitable buffersinclude, for example, TAPS(3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Bicine(N,N-bis(2-hydroxyethyl)glycine), IRIS (tris(hydroxymethyl)methylamine),ACES (N-(2-Acetamido)-2-aminoethanesulfonic acid), Tricine(N-tris(hydroxymethyl)methylglycine), HEPES4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TES(2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)), and MES(2-(N-morpholino)ethanesulfonic acid).

The pH of the reaction can influence the kinetics of the polymerasereaction, and can be used as one of the polymerase reaction conditionsto obtain a reaction exhibiting two slow-step kinetics. The pH can beadjusted to a value that produces a two slow-step reaction mechanism.The pH is generally between about 6 and about 9. In some embodiments,the pH is between about 6.5 and about 8.0. In other embodiments, the pHis between about 6.5 and 7.5. In particular embodiments, the pH isselected from about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or7.5.

The temperature of the reaction can be adjusted to ensure that therelative rates of the reactions are occurring in the appropriate range.The reaction temperature may depend upon the type of polymerase orselective cleaving activity employed. The temperatures used herein arealso contemplated to manipulate and control the hydrogen bonding betweentwo bases as well as the bases' interaction with the water in thereaction mixture, thereby controlling the solubility of the reactioncomponents. The temperature with also affect the vinding efficiency ofthe non-covalently attached quenchers. In particular embodiments,temperatures between 15° C. and 90° C., between 20° C. and 50° C.,between 20° C. and 40° C., or between 20° C. and 30° C. can be used.

In some embodiments, additives can be added to the reaction mixture thatwill influence the kinetics of the reaction. In some cases, theadditives can interact with the active site of the enzyme, acting forexample as competitive inhibitors. In some cases, additives can interactwith portions of the enzyme away from the active site in a manner thatwill influence the kinetics of the reaction. Additives that caninfluence the kinetics include, for example, competitive but otherwiseunreactive substrates or inhibitors in analytical reactions to modulatethe rate of reaction as described in U.S. Utility Pat. No. 8,252,911,the full disclosure of which is incorporated herein by reference in itsentirety for all purposes.

As another example, an isotope such as deuterium can be added toinfluence the rate of one or more step in the polymerase reaction. Insome cases, deuterium can be used to slow one or more steps in thepolymerase reaction due to the deuterium isotope effect. By altering thekinetics of steps of the polymerase reaction, in some instances two slowstep kinetics, as described herein, can be achieved. The deuteriumisotope effect can be used, for example, to control the rate ofincorporation of nucleotide, e.g., by slowing the incorporation rate.Isotopes other than deuterium can also be employed, for example,isotopes of carbon (e.g. ¹³C), nitrogen, oxygen, sulfur, or phosphorous.

As yet another example, additives that can be used to control thekinetics of the polymerase reaction include the addition of organicsolvents. The solvent additives are generally water soluble organicsolvents. The solvents need not be soluble at all concentrations, butare generally soluble at the amounts used to control the kinetics of thepolymerase reaction. While not being bound by theory, it is believedthat the solvents can influence the three dimensional conformation ofthe polymerase enzyme which can affect the rates of the various steps inthe polymerase reaction. For example, the solvents can affect stepsinvolving conformational changes such as the isomerization steps. Addedsolvents can also affect, and in some cases slow, the translocationstep. In some cases, the solvents act by influencing hydrogen bondinginteractions.

The water miscible organic solvents that can be used to control therates of one or more steps of the polymerase reaction in single moleculesequencing include, e.g., alcohols, amines, amides, nitriles,sulfoxides, ethers, and esters and small molecules having more than oneof these functional groups. Exemplary solvents include alcohols such asmethanol, ethanol, propanol, isopropanol, glycerol, and small alcohols.The alcohols can have one, two, three, or more alcohol groups. Exemplarysolvents also include small molecule ethers such as tetrahydrofuran(THF) and dioxane, dimethylacetamide (DMA), dimethylsulfoxide (DMSO),dimethylformamide (DMF), and acetonitrile.

The water miscible organic solvent can be present in any amountsufficient to control the kinetics of the polymerase reaction. Thesolvents are generally added in an amount less than 40% of the solventweight by weight or volume by volume. In some embodiments the solventsare added between about 0.1% and 30%, between about 1% and about 20%,between about 2% and about 15%, and between about 5% and 12%. Theeffective amount for controlling the kinetics can be determined by themethods described herein and those known in the art.

Another aspect of controlling the polymerase reaction conditions relatesto the selection of the type, level, and relative amounts of cofactors.For example, during the course of the polymerase reaction, divalentmetal co-factors, such as magnesium or manganese, will interact with theenzyme-substrate complex, playing a structural role in the definition ofthe active site. For a discussion of metal co-factor interactions inpolymerase reactions, see, for example, Arndt, et al., Biochemistry(2001) 40:5368-5375. Suitable conditions include those described in U.S.Pat. No. 8,257,954, incorporated herein by reference in its entirety forall purposes.

In a particular embodiment of the invention methods, the rate andfidelity of the polymerase reaction is controlled by adjusting theconcentrations of the dNTP nucleotide analogs such that the polymeraseoperates in near ideal conditions in terms of parameters such assubstrate concentration, amount of optical excitation, level of chemicalmodification. Therefore, the polymerase enzyme is contemplated herein toreach its maximum read-lengths, e.g., approximately in the tens ofthousands of base pairs, similar to the DNA synthesis lengths achievedin natural settings. This reduces device complexity and increasesenzymatic sensitivity and specificity leading to low error-rates andthus low coverage. This not only reduces the cost of the device as wellas cost per genome, but also makes applications such assingle-nucleotide polymerism detection, structural variation, and genomeassembly possible in a very compact system.

In another embodiment, as set forth above, because the quenching enzyme(e.g., ATP sulfurylase) and polymerase rates can vary significantlydepending on the type and source of the enzymes, the rate of quenchingachieved by the ATP sulfurylase reaction employed herein can be adjustedseparately by adjusting reaction conditions such as ATP sulfurylaseconcentration.

The invention includes systems for sequencing of nucleic acid templates.The systems provide for concurrently sequencing a plurality of nucleicacid templates. The system can incorporate all of the reagents andmethods described herein, and provides the instrumentation required forcontaining the sample, illuminating the sample with excitation light,detecting light emitted from the sample during sequencing to produceintensity versus time data from the labeled leaving groups cleaved fromthe nucleotide analogs as they are incorporated by the polymerase ontoits congnate template dna and from the labeled leaving groups, e.g.,fluorophore-labeled pyrophosphate, determining the sequence of atemplate using the sequential intensity versus time data.

As used herein, the phrase “detecting light” refers to well-knownmethods for detecting, for example, fluorescence emitted fromfluorophore labels when such labels are in their excitation stateemitting their respective signal.

The system for sequencing generally comprises a substrate having aplurality of single polymerase enzymes, single templates, or singleprimers within, for example, a unique droplet, or the like. In the caseof highly processive enzyme polymerase reactions, each comprising apolymerase enzyme, a nucleic acid template, and a primer are uniquelyconfined such that their signals can be assigned to the respectivenucleotide as gene synthesis occurs. The sequencing reagents generallyinclude two or more types of nucleotide analogs, preferably fournucleotide analogs corresponding dATP, dATP, dAGP and dCTP, eachnucleotide analog labeled with a different label. The polymerasesequentially adds nucleotides or nucleotide analogs to the growingstrand, which extends from the primer. Each added nucleotide ornucleotide analog is complementary to the corresponding base on thetemplate nucleic acid, such that the portion of the growing strand thatis produced is complementary to the template.

The system comprises illumination optics for illuminating the labeledleaving groups from the respective dNTPs as they are incorporated intothe template strand, e.g., labeled pyrophosphates. The illuminationoptics illuminate the labeled leaving groups in a wavelength range thatwill excite the labels on the cleaved pyrophosphate (no longer quenchedby the nucleobase).

The system further comprises detection optics for observing signals fromthe labeled leaving groups cleaved from the respective dNTP during thepolymerase enzyme mediated addition to the template strand. Thedetection optics observe a plurality of single molecule polymerasesequencing reactions concurrently, observing the nucleotide ornucleotide analog additions for each of them via the labeled leavinggroup (e.g., fluorophore-labeled pyrophosphate; PP). For each of theobserved single molecule polymerase sequencing reactions, the detectionoptics concurrently observe the signals from each of the labeled leavinggroups that are indicative of the respective unquenchedfluorophore-labeled corresponding to a respective dNTP, until eachrespective signal is quenched by the quenching enzyme (e.g., ATPsulfurylase).

The system also comprises a computer configured to determine the type ofnucleotide analog that is added to the growing strand using the observedsignal from the respective leaving group; whereby observed signals fromthe labeled leaving group are used to indicate whether a type ofnucleotide or nucleotide analog is incorporated into the growing strand.The computer generally receives information regarding the observedsignals from the detection optics in the form of signal data. Thecomputer stores, processes, and interprets the signal data, using thesignal data in order to produce a sequence of base calls. The base callsrepresent the computers estimate of the sequence of the template fromthe signal data received combined with other information given to thecomputer to assist in the sequence determination.

Optical illumination and detections systems which can be used with thepresent invention are described, for example in U.S. Pat. Nos.8,802,424; 7,714,303; and 7,820,983, each of which are incorporatedherein by reference in their entirety for all purposes.

Computers for use in carrying out the processes of the invention canrange from personal computers such as PC or Macintosh.R™. type computersrunning Intel Pentium or DuoCore processors, to workstations, laboratoryequipment, or high speed servers, running UNIX, LINUX, Windows.R™., orother systems, Logic processing of the invention may be performedentirely by general purposes logic processors (such as CPU's) executingsoftware and/or firmware logic instructions; or entirely by specialpurposes logic processing circuits (such as ASICs) incorporated intolaboratory or diagnostic systems or camera systems which may alsoinclude software or firmware elements; or by a combination of generalpurpose and special purpose logic circuits. Data formats for the signaldata may comprise any convenient format, including digital image baseddata formats, such as JPEG, GIF, BMP, TIFF, or other sequencing specificformats including “fastq” or the “qseq” format (Illumina); while videobased formats, such as avi, mpeg, mov, rmv, or other video formats maybe employed. The software processes of the invention may generally beprogrammed in a variety of programming languages including, e.g.,Matlab, C, C++, C#, NET, Visual Basic, Python, JAVA, CGI, and the like.

In some embodiments of the methods and systems of the invention, opticalconfinements are used to enhance the ability to concurrently observemultiple single molecule polymerase sequencing reactions simultaneously.In general, optical confinements are disposed upon a substrate and usedto provide electromagnetic radiation to or derive such radiation fromonly very small spaces or volumes. Such optical confinements maycomprise structural confinements, e.g., wells, recesses, conduits, orthe like, or they may comprise optical processes in conjunction withother components, to provide illumination to or derive emitted radiationfrom only very small volumes. Examples of such optical confinementsinclude systems that utilize, e.g., total internal reflection (TIR)based optical systems whereby light is directed through a transparentportion of the substrate at an angle that yields total internalreflection within the substrate.

In a particular embodiment, a preferred optical confinement is amicro-droplet which can contain and individual sequencing reaction setforth herein. For example, the sequencing mixture reaction ingredientscan be split in a way that each micro-droplet contains one polymeraseand one template nucleic acid whereby each signal detection unit isfocused on a single micro-droplet. It is contemplate herein that eachmicro-droplet is a single molecule reaction cell containing individualsingle molecule sequencing reactions. The micro-droplet reaction cell isalso advantageously useful in the invention sequencing methods to act asmicro-lenses to focus light on the reaction and to the respective signaldetection unit.

The substrates of the invention are generally rigid, and often planar,but need not be either. Where the substrate comprises an array ofoptical confinements, the substrate will generally be of a size andshape that can interface with optical instrumentation to allow for theillumination and for the measurement of light from the opticalconfinements. Typically, the substrate will also be configured to beheld in contact with liquid media, for instance containing reagents andsubstrates and/or labeled components, such as the fluorophore-labeledpyrophosphates, for optical measurements.

Where the substrates comprise arrays of optical confinements, the arraysmay comprise a single row or a plurality of rows of optical confinementon the surface of a substrate, where when a plurality of lanes arepresent, the number of lanes will usually be at least 2, more commonlymore than 10, and more commonly more than 100. The subject array ofoptical confinements may align horizontally or diagonally long thex-axis or the y-axis of the substrate. The individual confinements canbe arrayed in any format across or over the surface of the substrate,such as in rows and columns so as to form a grid, or to form a circular,elliptical, oval, conical, rectangular, triangular, or polyhedralpattern. To minimize the nearest-neighbor distance between adjacentoptical confinements, a hexagonal array is sometimes preferred.

The array of optical confinements may be incorporated into a structurethat provides for ease of analysis, high throughput, or otheradvantages, such as in a microtiter plate and the like. Such setup isalso referred to herein as an “array of arrays.” For example, thesubject arrays can be incorporated into another array such as microtiterplate wherein each micro well of the plate contains a subject array ofoptical confinements.

In accordance with the invention, arrays of confinements (e.g., reactioncells, micro-droplets, and the like) are provided in arrays of more than100, more than 1000, more than 10,000, more that 100,000, or more than1,000,000 separate reaction cells (such as a micro-droplet or the like)on a single substrate. In addition, the reaction cell arrays aretypically comprised in a relatively high density on the surface of thesubstrate. Such high density typically includes reaction cells presentat a density of greater than 10 reaction cells per mm², preferably,greater than 100 reaction cells per mm² of substrate surface area, andmore preferably, greater than 500 or even 1000 reaction cells per mm²and in many cases up to or greater than 100,000 reaction cells per mmmm². Although in many cases, the reaction cells in the array are spacedin a regular pattern, e.g., in 2, 5, 10, 25, 50 or 100 or more rowsand/or columns of regularly spaced reaction cells in a given array, incertain preferred cases, there are advantages to providing theorganization of reaction cells in an array deviating from a standard rowand/or column format. In preferred aspects, the substrates include asthe particular reaction cell micro-droplets as the optical confinementsto define the discrete single molecule sequencing reaction regions onthe substrate.

The overall size of the array of optical confinements can generallyrange from a few nanometers to a few millimeters in thickness, and froma few millimeters to 50 centimeters in width and/or length. Arrays mayhave an overall size of about few hundred microns to a few millimetersin thickness and may have any width or length depending on the number ofoptical confinements desired.

The spacing between the individual confinements can be adjusted tosupport the particular application in which the subject array is to beemployed. For instance, if the intended application requires adark-field illumination of the array without or with a low level ofdiffractive scattering of incident wavelength from the opticalconfinements, then the individual confinements may be placed close toeach other relative to the incident wavelength.

The individual confinement in the array can provide an effectiveobservation volume less than about 1000 zeptoliters, less than about900, less than about 200, less than about 80, less than about 10zeptoliters. Where desired, an effective observation volume less than 1zeptoliter can be provided. In a preferred aspect, the individualconfinement yields an effective observation volume that permitsresolution of individual molecules, such as enzymes, present at or neara physiologically relevant concentration. The physiologically relevantconcentrations for many biochemical reactions range from micro-molar tomillimolar because most of the enzymes have their Michaelis constants inthese ranges. Accordingly, preferred array of optical confinements hasan effective observation volume for detecting individual moleculespresent at a concentration higher than about 1 micromolar (uM), or morepreferably higher than 50 uM, or even higher than 100 uM. In particularembodiments, typical microdroplet sizes range from 10 micrometers to 200micrometers, and thus typical microdroplet volumes are around 5picoliters to 20 nanoliters.

In the context of chemical or biochemical analyses within opticalconfinements, it is generally desirable to ensure that the reactions ofinterest are taking place within the optically interrogated portions ofthe confinement, at a minimum, and preferably such that only thereactions of a single molecule polymerase sequencing reaction isoccurring within an interrogated portion of an individual confinement(e.g., within a micro-droplet, or the like). A number of methods maygenerally be used to provide individual molecules within the observationvolume. A variety of these are described in U.S. Pat. No. 7,763,423,incorporated herein by reference in its entirety for all purposes, whichdescribes, inter alia, modified surfaces that are designed to immobilizeindividual molecules to the surface at a desired density, such thatapproximately one, two, three or some other select number of moleculeswould be expected to fall within a given observation volume. Typically,such methods utilize dilution techniques to provide relatively lowdensities of coupling groups on a surface, either through dilution ofsuch groups on the surface or dilution of intermediate or final couplinggroups that interact with the molecules of interest, or combinations ofthese. Also contemplated herein is the use of these dilution techniquesfor providing one, two, three or some other select number of singlemolecule sequencing reactions to fall within a given observation volumewithout being immobilized to a surface, such as would occur in themicro-droplet reaction cell contemplated herein for optical confinement.In a particular embodiment, the dilution techniques are utilized toprovide a one single molecule sequencing reaction in a micro-droplet foruse in the invention sequencing method.

The systems and methods of the inventions can result in improvedsequence determination and improved base calling by monitoring thesignal from the labeled leaving groups of the nucleotide analogs, e.g.,a polyphosphate label, using systems well-known in the art. In general,signal data is received by the processor. The information received bythe processor can come directly from the detection optics, or the signalfrom the detection optics can be treated by other processors beforebeing received by the processor. A number of initial calibrationoperations may be applied. Some of these initial calibration steps maybe performed just once at the beginning of a run or on a more continuousbasis during the run. These initial calibration steps can include suchthings as centroid determination, alignment, gridding, drift correction,initial background subtraction, noise parameter adjustment, frame-rateadjustment, etc. Some of these initial calibration steps, such asbinning, may involve communication from the processor back to thedetector/camera, as discussed further below.

Generally, some type of spectral trace determination, spectral traceextraction, or spectral filters are applied to the initial signal data.Some or all of these filtration steps may optionally be carried out at alater point in the process, e.g., after the pulse identification step.The spectral trace extraction/spectral filters may include a number ofnoise reduction and other filters as is well-known in the art. Spectraltrace determination is performed at this stage for many of the examplesystems discussed herein because the initial signal data received arethe light levels, or photon counts, captured by a series of adjacentpixel detectors. For example, in one example system, pixels (orintensity levels) from positions are captured for an individualwave-guide at each frame. Light of different frequencies or spectrumwill fall on more than one of the positions and there is generally someoverlap and possibly substantial overlap. According to specificembodiments of the invention, spectral trace extraction may be performedusing various type of analyses, as discussed below, that provide thehighest signal-to-noise ratio for each spectral trace.

As an alternative to a spectral trace determination, methods of theinvention may also analyze a single signal derived from the intensitylevels at the multiple pixel positions (this may be referred to as asummed spectral signal or a gray-scale spectral signal or an intensitylevel signal). In many situations, it has been found that spectralextraction, however, provides better SNR (signal to noise ratio) andtherefore pulse detection when extracted spectral traces are analyzedfor pulses somewhat separately. In further embodiments, a methodaccording to the invention may analyze the multiple captured pixel datausing a statistical model such as a Hidden Markov Model. In theinvention sequencing methods and systems provided herein, determiningmultiple (e.g., four) spectral traces from the initial signal data is apreferred method.

Whether the signal from the labeled leaving groups can be categorized asa significant signal pulse or event is determined. In some examplesystems, because of the small number of photons available for detectionand because of the speed of detection, various statistical analysistechniques may be performed in determining whether a significant pulsehas been detected.

If the signal is identified as a significant pulse or signal event, afurther optional spectral profile comparison may be performed to verifythe spectral assignment. This spectral profile comparison is optional inembodiments where spectral traces are determined prior to or duringpulse identification. Once a color is assigned to a given incorporationsignal (e.g., a fluorophore-labeled dNTP\), that assignment is used tocall either the respective base incorporated, or its complement in thetemplate sequence. In order to make this determination, the signalscoming from the channel corresponding to the labeled leaving group areused to assess whether a pulse from a nucleotide label corresponds to anincorporation event. The compilation of called bases is then subjectedto additional processing to provide linear sequence information, e.g.,the successive sequence of nucleotides in the template sequence,assemble sequence fragments into longer contigs, or the like.

As noted above, the signal data is input into the processing system,e.g., an appropriately programmed computer or other processor. Signaldata may input directly from a detection system, e.g., for real timesignal processing, or it may be input from a signal data storage file ordatabase. In some cases, e.g., where one is seeking immediate feedbackon the performance of the detection system, adjusting detection or otherexperimental parameters, real-time signal processing will be employed.In some embodiments, signal data is stored from the detection system inan appropriate file or database and is subject to processing in postreaction or non-real time fashion.

The signal data used in conjunction with the present invention may be ina variety of forms. For example, the data may be numerical datarepresenting intensity values for optical signals received at a givendetector or detection point of an array based detector. Signal data maycomprise image data from an imaging detector, such as a CCD, EMCCD, ICCDor CMOS sensor. In particular embodiments, for detecting low numbers ofphotons from single molecules, the use of a photomultiplier tube (PMT)and/or a photon counter unit is contemplated for use in the inventionmethods. In either event, signal data used according to specificembodiments of the invention generally includes both intensity levelinformation and spectral information. In the context of separatedetector elements, such spectral information will generally includesidentification of the location or position of the detector portion(e.g., a pixel) upon which an intensity is detected. In the context ofimage data, the spectral image data will typically be the data derivedfrom the image data that correlates with the calibrated spectral imagedata for the imaging system and detector when the system includesspectral resolution of overall signals. The spectral data may beobtained from the image data that is extracted from the detector, oralternatively, the derivation of spectral data may occur on the detectorsuch that spectral data will be extracted from the detector.

For the sequencing methods described above, there will be a certainamount of optical signal that is detected by the detection system thatis not the result of a signal from an incorporation event. Such signalwill represent “noise” in the system, and may derive from a number ofsources that may be internal to the monitored reaction, internal to thedetection system and/or external to all of the above. The practice ofthe present invention advantageously reduces these overall sources ofnoise typically present in prior art methods. Examples of prior artnoise internal to the reaction that is advantageously reduced inaccordance with the present invention includes, e.g.: presence offluorescent labels that are not associated with a detection event, e.g.,liberated labels, labels associated with unincorporated bases indiffused in solution, bases associated with the complex but notincorporated; presence of multiple complexes in an individualobservation volume or region; non-specific adsorption of dyes ornucleotides to a substrate or enzyme complex within an observationvolume; contaminated nucleotide analogs, e.g., contaminated with otherfluorescent components; other reaction components that may be weaklyfluorescent; spectrally shifting dye components, e.g., as a result ofreaction conditions; and the like. The controlled use of fluorescentsignal detection and information from the fluorescent label on theleaving group of the respective dNTP that then becomes quenched prior tothe incorporation of the next nucleotide analog advantageously providesa way of reducing or eliminating sources of noise, thereby improving thesignal to noise of the system, and improving the quality of the basecalls and associated sequence determination.

Sources of noise internal to the detection system, but outside of thereaction mixture can include, e.g., reflected excitation radiation thatbleeds through the filtering optics; scattered excitation or fluorescentradiation from the substrate or any of the optical components; spatialcross-talk of adjacent signal sources; auto-fluorescence of any or allof the optical components of the system; read noise from the detector,e.g., CCDs, gain register noise, e.g., for EMCCD cameras, and the like.Other system derived noise contributions can come from data processingissues, such as background correction errors, focus drift errors,autofocus errors, pulse frequency resolution, alignment errors, and thelike. Still other noise contributions can derive from sources outside ofthe overall system, including ambient light interference, dust, and thelike.

These noise components contribute to the background photons underlyingany signal pulses that may be associated with an incorporation event. Assuch, the noise level will typically form the limit against which anysignal pulses may be determined to be statistically significant.

Identification of noise contribution to overall signal data may becarried out by a number of methods, including, for example, signalmonitoring in the absence of the reaction of interest, where any signaldata is determined to be irrelevant. Alternatively, and preferably, abaseline signal is estimated and subtracted from the signal data that isproduced by the system, so that the noise measurement is made upon andcontemporaneously with the measurements on the reaction of interest.Generation and application of the baseline may be carried out by anumber of means, which are described in greater detail below.

In accordance with the present invention, signal processing methodsdistinguish between noise, as broadly applied to all non-significantpulse based signal events, and significant signal pulses that may, witha reasonable degree of confidence, be considered to be associated with,and thus can be tentatively identified as, an incorporation event. Inthe context of the present invention, a signal event is first classifiedas to whether it constitutes a significant signal pulse based uponwhether such signal event meets any of a number of different pulsecriteria. Once identified or classified as a significant pulse, thesignal pulse may be further assessed to determine whether the signalpulse constitutes an incorporation event and may be called as aparticular incorporated base. As will be appreciated, the basis forcalling a particular signal event as a significant pulse, and ultimatelyas an incorporation event, will be subject to a certain amount of error,based upon a variety of parameters as generally set forth herein. Assuch, it will be appreciated that the aspects of the invention thatinvolve classification of signal data as a pulse, and ultimately as anincorporation event or an identified base, are subject to the same orsimilar errors, and such nomenclature is used for purposes of discussionand as an indication that it is expected with a certain degree ofconfidence that the base called is the correct base in the sequence, andnot as an indication of absolute certainty that the base called isactually the base in a given position in a given sequence.

One such signal pulse criterion is the ratio of the signals associatedwith the signal event in question to the level of all background noise(“signal to noise ratio” or “SNR”), which provides a measure of theconfidence or statistical significance with which one can classify asignal event as a significant signal pulse. In distinguishing asignificant pulse signal from systematic or other noise components, thesignal generally must exceed a signal threshold level in one or more ofa number of metrics, including for example, signal intensity, signalduration, temporal signal pulse shape, pulse spacing, and pulse spectralcharacteristics.

By way of a simplified example, signal data may be input into theprocessing system. If the signal data exceeds a signal threshold valuein one or more of signal intensity and signal duration, it may be deemeda significant pulse signal. Similarly, if additional metrics areemployed as thresholds, the signal may be compared against such metricsin identifying a particular signal event as a significant pulse. As willbe appreciated, this comparison will typically involve at least one ofthe foregoing metrics, and preferably at least two such thresholds, andin many cases three or all four of the foregoing thresholds inidentifying significant pulses.

Signal threshold values, whether in terms of signal intensity, signalduration, pulse shape, spacing or pulse spectral characteristics, or acombination of these, will generally be determined based upon expectedsignal profiles from prior experimental data, although in some cases,such thresholds may be identified from a percentage of overall signaldata, where statistical evaluation indicates that such thresholding isappropriate. In particular, in some cases, a threshold signal intensityand/or signal duration may be set to exclude all but a certain fractionor percentage of the overall signal data, allowing a real-time settingof a threshold. Again, however, identification of the threshold level,in terms of percentage or absolute signal values, will generallycorrelate with previous experimental results. In alternative aspects,the signal thresholds may be determined in the context of a givenevaluation. In particular, for example, a pulse intensity threshold maybe based upon an absolute signal intensity, but such threshold would nottake into account variations in signal background levels, e.g., throughreagent diffusion, that might impact the threshold used, particularly incases where the signal is relatively weak compared to the backgroundlevel. As such, in certain aspects, the methods of the inventiondetermine the background fluorescence of the particular reaction inquestion, which is relatively small because the contribution of freelydiffusing dyes or dye labeled analogs into a micro-droplet is minimal ornon-existent, and sets the signal threshold above that actual backgroundby the desired level, e.g., as a ratio of pulse intensity to backgroundfluorophore diffusion, or by statistical methods, e.g., 5 sigma, or thelike. By correcting for the actual reaction background, such as theminimal fluorophore diffusion background, the threshold is automaticallycalibrated against influences of variations in dye concentration, laserpower, or the like. By reaction background is meant the level ofbackground signal specifically associated with the reaction of interestand that would be expected to vary depending upon reaction conditions,as opposed to systemic contributions to background, e.g.,autofluorescence of system or substrate components, laser bleedthrough,or the like.

In particularly preferred aspects that rely upon real-time detection ofincorporation events, identification of a significant signal pulse mayrely upon a signal profile that traverses thresholds in both signalintensity and signal duration. For example, when a signal is detectedthat crosses a lower intensity threshold in an increasing direction,ensuing signal data from the same set of detection elements, e.g.,pixels, are monitored until the signal intensity crosses the same or adifferent intensity threshold in the decreasing direction. Once a peakof appropriate intensity is detected, the duration of the period duringwhich it exceeded the intensity threshold or thresholds is comparedagainst a duration threshold. Where a peak comprises a sufficientlyintense signal of sufficient duration, it is called as a significantsignal pulse.

In addition to, or as an alternative to using the intensity and durationthresholds, pulse classification may employ a number of other signalparameters in classifying pulses as significant. Such signal parametersinclude, e.g., pulse shape, spectral profile of the signal, e.g., pulsespectral centroid, pulse height, pulse diffusion ratio, pulse spacing,total signal levels, and the like.

Either following or prior to identification of a significant signalpulse, signal data may be correlated to a particular signal type. In thecontext of the optical detection schemes used in conjunction with theinvention, this typically denotes a particular spectral profile of thesignal giving rise to the signal data. In particular, the opticaldetection systems used in conjunction with the methods and processes ofthe invention are generally configured to receive optical signals thathave distinguishable spectral profiles, where each spectrallydistinguishable signal profile may generally be correlated to adifferent reaction event. In the case of nucleic acid sequencing, forexample, each spectrally distinguishable signal may be correlated orindicative of a specific nucleotide incorporated or present at a givenposition of a nucleic acid sequence. Consequently, the detection systemsinclude optical trains that receive such signals and separate thesignals based upon their spectra. The different signals are thendirected to different detectors, to different locations on a singlearray based detector, or are differentially imaged upon the same imagingdetector (See, e.g., U.S. Pat. No. 7,805,081, which is incorporatedherein by reference in its entirety for all purposes).

In the case of systems that employ different detectors for differentsignal spectra, assignment of a signal type (for ease of discussion,referred to hereafter as “color classification” or “spectralclassification”) to a given signal is a matter of correlating the signalpulse with the detector from which the data derived. In particular,where each separated signal component is detected by a discretedetector, a signal's detection by that detector is indicative of thesignal classifying as the requisite color.

In preferred aspects, however, the detection systems used in conjunctionwith the invention utilize an imaging detector upon which all or atleast several of the different spectral components of the overall signalare imaged in a manner that allows distinction between differentspectral components. Thus, multiple signal components are directed tothe same overall detector, but may be incident upon wholly or partlydifferent regions of the detector, e.g., imaged upon different sets ofpixels in an imaging detector, and give rise to distinguishable spectralimages (and associated image data). As used herein, spectra or spectralimage generally indicates a pixel image or frame (optionally datareduced to one dimension) that has multiple intensities caused by thespectral spread of an optical signal received from a reaction location.

In its simplest form, it will be understood that assignment of color toa signal event incident upon a group of contiguous detection elements orpixels in the detector would be accomplished in a similar fashion asthat set forth for separate detectors. In particular, the position ofthe group of pixels upon which the signal was imaged, and from which thesignal data is derived, is indicative of the color of the signalcomponent. In particularly preferred aspects, however, spatialseparation of the signal components may not be perfect, such thatsignals of differing colors are imaged on overlapping sets of pixels. Assuch, signal identification will generally be based upon the aggregateidentity of multiple pixels (or overall image of the signal component)upon which a signal was incident.

Once a particular signal is identified as a significant pulse and isassigned a particular spectrum, the spectrally assigned pulse may befurther assessed to determine whether the pulse can be called anincorporation event and, as a result, call the base incorporated in thenascent strand, or its complement in the template sequence. Signals fromthe labeled leaving group (e.g., fluorophore labeled pyrophosphate) areused to identify which base should be called. As set forth above, in oneembodiment, by using two quenchers per nucleotide analog, such as thenucleobase of the nucleotide and a non-covalently attached quencher, aset of characteristic signals are produced which can be correlated withhigh confidence to an incorporation event.

In addition, calling of bases from color assigned pulse data willtypically employ tests that again identify the confidence level withwhich a base is called. Typically, such tests will take into account thedata environment in which a signal was received, including a number ofthe same data parameters used in identifying significant pulses. Forexample, such tests may include considerations of background signallevels, adjacent pulse signal parameters (spacing, intensity, duration,etc.), spectral image resolution, and a variety of other parameters.Such data may be used to assign a score to a given base call for a colorassigned signal pulse, where such scores are correlative of aprobability that the base called is incorrect, e.g., 1 in 100 (99%accurate), 1 in 1000 (99.9% accurate), 1 in 10,000 (99.99% accurate), 1in 100,000 (99.999% accurate), or even greater. Similar to PHRED orsimilar type scoring for chromatographically derived sequence data, suchscores may be used to provide an indication of accuracy for sequencingdata and/or filter out sequence information of insufficient accuracy.

Once a base is called with sufficient accuracy, subsequent bases calledin the same sequencing run, and in the same primer extension reaction,may then be appended to each previously called base to provide asequence of bases in the overall sequence of the template or nascentstrand. Iterative processing and further data processing can be used tofill in any blanks, correct any erroneously called bases, or the likefor a given sequence.

Analysis of sequencing-by-incorporation-reactions on an array ofreaction locations according to specific embodiments of the inventioncan be conducted as illustrated graphically in FIG. 13 of U.S. Pat. No.9,447,464, incorporated by reference in its entirety for all purposes).For example, data captured by a camera is represented as a movie, whichis also a time sequence of spectra. Spectral calibration templates areused to extract traces from the spectra. Pulses identified in the tracesare then used to return to the spectra data and from that data produce atemporally averaged pulse spectrum for each pulse, such pulse spectrawill include spectra for events relating to enzyme conformationalchanges. The spectral calibration templates are then also used toclassify pulse spectrum to a particular base. Base classifications andpulse and trace metrics are then stored or passed to other logic forfurther analysis. The downstream analysis will include using theinformation from enzyme conformational changes to assist in thedetermination of incorporation events for base calling. Further basecalling and sequence determination methods for use in the invention caninclude those described in, for esample, U.S. Pat. No. 8,182,993, whichis incorporated herein by reference in its entirety for all purposes.

EXAMPLES Single Molecule Sequencing

Prior to undergoing a single molecule sequencing reaction, therespective fluorophores are attached to the terminal phosphate of itscorresponding dNTP for each of dATP, dTTP, dGTP and dCTP, whichfluorophore is quenched by the nucleobase while it is attached to thedNTP. Each nucleobase has a different quenching capability for thedifferent fluorophores (FIG. 2.A). During the single molecule sequencingreaction, upon interaction with the DNA polymerase, while the DNApolymerase binds the dNTP nucleotide analog to the template strand, itcleaves off a pyrophosphate that has the signaling fluorophore attachedthereto (FIG. 2.B).

As a result of dNTP interacting with the DNA polymerase, fluorescencelight is generated upon cleavage of labeled pyrophosphate generating afluorescence signal corresponding to the color of the fluorophoreselected for the particular dNTP. There is a different fluorophore foreach dNTP base (A, T, G, C) (FIG. 2.C). The respective fluorescent lightis the detected prior to the light becoming quenched.

Once released and its fluorescent light has been detected, the labeledpyrophosphate (PP,) interacts with ATP sulfurylase, which binds thelabeled pyrophosphate to adenosine 5′-phosphosulfate (APS) resulting inthe quenching of the fluorophore by adenine upon binding. The quenchingof the fluorophore labels on the pyrophosphates substantially eliminatesall background fluorescent signals in the sequencing mixture (FIG. 2.D).

This dNTP incorporation process is repeated until the desired nucleicacid read-length has been achieved.

While the present embodiments have been particularly shown and describedwith reference to example embodiments herein, it will be understood bythose of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present embodiments as defined by the following claims. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of the present invention and are covered by thefollowing claims. The contents of all non-patent literaturepublications, patents, and patent applications cited throughout thisapplication are hereby incorporated by reference in their entirety forall purposes. The appropriate components, processes, and methods ofthose patents, applications and other documents may be selected for thepresent invention and embodiments thereof.

1. A method for sequencing a nucleic acid template comprising: providinga sequencing mixture comprising (i) a polymerase enzyme, (ii) a templatenucleic acid, and (iii) a polymerase reagent solution having thecomponents for carrying out template directed synthesis of a growingnucleic acid strand, wherein said polymerase reagent solution includes acomponent for a requenching reaction and a plurality of types ofnucleotide analogs; wherein each type of nucleotide analog has a labeledleaving group that is cleavable by the polymerase, and each type ofnucleotide analog has a different label, wherein the labeled leavinggroup is cleaved upon polymerase-dependent binding of a respectivenucleotide analog to the template strand; carrying out nucleic acidsynthesis such that a plurality of nucleotide analogs are addedsequentially to the template whereby: a) a nucleotide analog associateswith the polymerase, b) the nucleotide analog is incorporated on thetemplate strand by the polymerase when the labeled leaving group on thatnucleotide analog is cleaved by the polymerase, wherein the labeledleaving group emits light upon cleavage, then c) the labeled leavinggroup on the nucleotide analog is quenched by the requenching reaction;and detecting light from the labels while nucleic acid synthesis isoccurring, and using light detected in the time between step b) when thelabelled leaving group is cleaved, and step c) in which the labeledleaving group is quenched, to determine a sequence of the templatenucleic acid.
 2. The method of claim 1, wherein the nucleotide analoghas been modified by a fluorophore attached to a terminal phosphate. 3.The method of claims 1, wherein the leaving group is a labelledpyrophosphate.
 4. The method of claims 3, wherein the pyrophosphate islabeled with a fluorophore.
 5. The method of claims 1, wherein each baseof a nucleotide is labeled with a unique fluorophore relative to otherbases.
 6. The method of claims 1, wherein the requenching reaction usesa quenching enzyme or Plasmonics.
 7. The method of claims 6, wherein thequenching enzyme is ATP sulfurylase.
 8. The method of claims 1, whereinrequenching is achieved by an adenosine nucleobase on APS.
 9. The methodof claims 1, wherein the polymerase enzyme is DNA polymerase.
 10. Themethod of claims 1, wherein types of nucleotide analogs comprise dATP,dTTP, dGTP, dCTP and dUTP.
 11. The method of claims 1, wherein therequenching reaction uses plasmonics.
 12. The method of claims 2,wherein the fluorophore is quenched by the nucleobase on the nucleotide.13. The method of claims 1, wherein a removable quencher molecule isattached to the nucleotide analog through electrostatic interaction orhydrogen bonding.
 14. The method of claim 13, wherein the removablequencher molecule is attached to the nucleotide analog at the nucleobaseor sugar.